Helicobacter pylori infection induces gastric precancerous lesions and persistent expression of Angpt2, Vegf-A and Tnf-A in a mouse model

Introduction Helicobacter pylori colonizes the gastric mucosa and induces chronic inflammation. Methods Using a mouse model of H. pylori-induced gastritis, we evaluated the mRNA and protein expression levels of proinflammatory and proangiogenic factors, as well as the histopathological changes in gastric mucosa in response to infection. Five- to six-week-old female C57BL/6N mice were challenged with H. pylori SS1 strain. Animals were euthanized after 5-, 10-, 20-, 30-, 40- and 50-weeks post infection. mRNA and protein expression of Angpt1, Angpt2, VegfA, Tnf-α, bacterial colonization, inflammatory response and gastric lesions were evaluated. Results A robust bacterial colonization was observed in 30 to 50 weeks-infected mice, which was accompanied by immune cell infiltration in the gastric mucosa. Compared to non-infected animals, H. pylori-colonized animals showed an upregulation in the expression of Tnf-A, Angpt2 and VegfA at the mRNA and protein levels. In contrast, Angpt1 mRNA and protein expression was downregulated in H. pylori-colonized mice. Conclusion Our data show that H. pylori infection induces the expression of Angpt2, Tnf-A and Vegf-A in murine gastric epithelium. This may contribute to the pathogenesis of H. pylori-associated gastritis, however the significance of this should be further addressed.


Introduction
Angiogenesis, the process of formation of new blood vessels from the pre-existing, involves proliferation, sprouting and migration of endothelial cells, as well as degradation of the basement membrane. After the re-establishment of cell junctions and coverage of pericytes, the newly formed vessels mature and remain quiescent (1). Importantly, endothelial cells start proliferating to initiate angiogenesis only after stimulation by several growth factors and inflammatory mediators, mainly members of vascular endothelial growth factor (VEGF) family and angiopoietins.
Angiogenesis and tumor promoting inflammation are closely interconnected and are crucial events during cancer progression and metastasis; in fact, they are both considered as hallmarks of cancer (2). Both tumor and stromal cells produce angiogenic factors that ultimately lead to endothelial cell proliferation. These cancerassociated endothelial cells cooperate in the perpetuation of inflammation, which reciprocally promotes angiogenesis by secretion of cytokines, proteases, growth and proangiogenic factors, thus creating a positive feedback loop (1,2). Aberrant vascular structures can also induce hypoxia, acidosis and DNA damage (3), which contribute to the establishment of a tumor-promoting microenvironment since very early in the carcinogenesis. This could be particularly relevant in cancers in which chronic and persistent inflammation is the main driving force for the malignant transformation, for example in gastric carcinogenesis.
VEGFA is the most potent and ubiquitous member of VEGF family; it is considered as the master inductor of physiological and tumor angiogenesis (4). Secreted by macrophages, lymphocytes, fibroblast and tumor cells, it promotes proliferation, migration and survival of endothelial cells, as well as expression of extracellular matrix proteases (5). Angiopoietin 1 (ANGPT1) and ANGPT2 bind to TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) receptor complex and a5b1 integrin in endothelial cells, thus activating signaling pathways that lead to blood and lymphatic vessel formation. ANGPT1 is secreted by pericytes and acts in a paracrine manner to keep homeostasis of the mature vasculature. ANGPT2 is mostly produced by endothelial cells, in which it is stored at the cytoplasmatic Weibel-Palade bodies, and released after stimulation, to act in an autocrine way (6). ANGPT2 plays a role as a vessel destabilizing agent that induces permeability and leads to dissociation of cell-cell contacts, allowing the sprouting of new vessels (1,7). The expression of ANGPT2 in normal tissues is low or absent, but it is upregulated in many cancers in which it is mainly expressed by tumor-associated macrophages (6). In humans, alternative splicing generates a smaller isoform, ANGPT2 443 , which has been reported as upregulated in tumor cell lines and human tumor tissue (8,9).
TNF-a is a key mediator in a host's response against gramnegative bacteria, such as H. pylori. It is produced by lymphocytes and macrophages and promotes leukocyte recruitment. In the context of angiogenesis, TNF-a primes endothelial cells for angiogenic sprouting by inducing a tip cell phenotype (10).
Helicobacter pylori colonization of the gastric mucosa induces chronic and persistent inflammation. This is associated with several clinical outcomes, such as peptic ulcer, MALT-lymphoma and gastric cancer (GC). The latter results from a stepwise cascade of preneoplastic lesions known as Correa's cascade (11,12). The persistent inflammation mounted in response to this bacterial infection is characterized by the infiltration in the gastric mucosa of neutrophils, lymphocytes and macrophages. Increased permeability of the endothelium is a pivotal event for the extravasation of immune cells, proteins and fluids (13).
Several reports indicate that H. pylori can induce the production of proangiogenic factors in vitro and in vivo (14)(15)(16)(17)(18). Specifically, the expression of HIF-1a, VEFGA and the density of CD31+ blood vessels is higher in H. pylori-positive patients, compared to negative persons (19,20). Those findings suggest that the bacterium is capable of inducing a proangiogenic response after colonizing its host. This could be of pivotal importance during gastric cancer initiation and development. However, the molecules and mechanisms by which H. pylori induces early neovascularization in gastric mucosa, and how this influences the precancerous series of events that precede GC is not completely understood.
The present study, we used a mouse model in order to characterize the expression kinetics of Tnf-A, Angpt1, Angpt2 and VegfA in the gastric mucosa in response to H. pylori infection and to explore the correlation to inflammation, vascularization and gastric pathology.

Animals
Thirty-nine C57BL/6N female mice certified as Helicobacter sppfree were acquired from Harlan Laboratories ® and housed at the Laboratorio de Ensayos Bioloǵicos (LEBi), University of Costa Rica, at standard temperature (25.5°C ± 1.20°C) and humidity (50-70%), and maintained on a 12-h light/dark cycle (lights off at 6:00 p.m). Water and food (LabDiet 5010.) were provided ad libitum. Mice were distributed into 7 experimental groups, each consisting of 5-7 animals per cage ( Figure 1) and were moved to an experimental section one week before they were inoculated with H. pylori. Experimental procedures and methods were carried out in accordance with the guidelines of the Costa Rican Ministry of Science and Technology and were approved by the Institutional Committee for Animal Care and Use of Animals (CICUA) of the University of Costa Rica (permission number CICUA-031-17).

Bacterial culture and inoculation
The inoculation was performed with SS1 strain (mouse-adapted cagA+, vacA+ strain with no functionality of the cag pathogenicity island; kindly donated by Dr. James Fox, MIT, USA). Bacteria were grown on Skirrow plates and incubated at 37°C for 5-7 days under microaerophilic conditions. The SS1 strain was harvested and mice were inoculated by oral gavage with a single dose of approximately 10 9 bacteria (CFU) in 0.2 mL of 0.9% NaCl. Control mice received 0.2 mL of the saline solution only. The bacteria used for inoculations were isolated from previously colonized mice from our bioterium.

Resection and processing of gastric tissue for histology
Infected groups were euthanized by cervical dislocation after 5-, 10-, 20-, 30-, 40-and 50-weeks post-infection (w.p.i.). The control group was euthanized at 50 w.p.i. Entire stomachs were opened along the greater curvature, washed with PBS, and cut longitudinally into four stripes, extending from the squamous forestomach through the duodenum. The stripes were used as follows: one for confirmation of bacterial colonization (culture), one was cut in halves for RNA and protein isolation, and two for immunohistochemistry assays.

Culture for confirmation of H. pylori colonization
The stripe was macerated with saline solution, vortexed 5 s, then 40 ml of the suspension was seeded in both Skirrow and agar-blood BHI plates and incubated at 37°C for 5-7 days under microaerophilic conditions.

Quantitative real time PCR
The stomach tissue was suspended in RNAlater (Invitrogen) until utilization. Total RNA was isolated with Trizol (Invitrogen) in accordance with the manufacturer's instructions. RNA from each sample was transcribed into cDNA using High-capacity cDNA reverse transcription kit (ThermoFisher Scientific). A total of 0,5 ml cDNA was used for qRT-PCR amplification using TaqMan Gene Expression Assays (Thermo Fisher Scientific) for Angpt1 (Mm00456503_m1), Angpt2 (Mm00545822_m1), Tnf-A (Mm00443258_m1) and VegfA (Mm00437306_m1) with Gapdh (Mm99999915_g1) as endogenous control, using the StepOne Plus thermal cycler (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95°C for 10 sec, 95°C for 5 sec, and 60°C for 30 sec, run for 40 cycles. Fold-difference for infected vs. non infected mice was estimated by the 2 -DDCt method.

Western blot analysis
Stomach tissues were macerated and lysed using lysis buffer solution (PBS 1X, 1% Triton X-100, 1% NP-40, proteases and phosphatases cocktail inhibitors, pH 7.4). Following centrifugation at 14 000 g for 30 min, the proteins were quantified using the Protein Assay Dye Reagent Concentrate (BioRad) and 50 µg of protein were separated by 10% SDS−PAGE, prior to being transferred onto a nitrocellulose membrane (BioRad). Following blocking with 5% fat −free milk in PBS buffer in 0.05% Tween 20 for 1 h at room temperature, the membranes were then separately incubated overnight at 4˚C with the following monoclonal antibodies: Rabbit ANGPT1 (1:500; Abcam ab102015), Rabbit ANGPT2 (1:500, Abcam ab8452) and Rabbit GAPDH (1:1000, Abcam, ab9485). The secondary antibodies were applied (dilution 1:10 000, horseradish peroxidase-conjugated anti-rabbit, Sigma-Aldrich A9169) at room temperature for 1 h. Labeled bands were detected by enhanced chemiluminescence (Clarity ECL, BioRad, USA) and analyzed by Chemidoc Imaging System (Bio-Rad). Protein levels were quantified using the Image Lab software, version 4.1 (Bio-Rad, USA).  Schematic view of the experimental groups of the mouse model of Helicobacter pylori SS1 infection used in the study. A total of 39 female C57BL/6N mice were divided into 7 groups (n per group indicated between parentheses), 6 of these groups were infected with H. pylori and one group received NaCl 0.9% only (control group, n=5). Mice were euthanized between 5-50 w.p.i. Control non-infected mice were sacrificed at 50 w.p.i. Created with BioRender.com.

H. pylori colonization, histopathology and immunohistochemistry evaluations
H. pylori colonization was evaluated according to the number of gastric glands containing bacteria and their density, as follows: 0: No observed bacteria; 1: Occasional pits and/or glands with individual bacteria; 2: frequent pits and/or glands with individual bacteria; 3, infrequent pits and/or gland with dense bacterial colonies; and 4, frequent pits and/or glands with dense bacterial colonies (22). Sections were stained with hematoxylin and eosin (H&E) for histopathological evaluations of the mouse gastric mucosa describing inflammation, infiltration, metaplasia, and anatomical localization of the lesions, using published guidelines (23): 0, no infiltration, 1, patchy or multifocal small islands of inflammatory cells in the mucosa and/or submucosa; 2, coalescing aggregates of inflammatory cells in submucosa or mucosa; 3, organizing nodules of lymphocytes and other inflammatory cells in submucosa and mucosa; 4, follicles and/or sheets of inflammatory cells extending into or through muscularis propria adventitia. Photomicrographs were captured using the bright field microscope Motic BA400 and Motic Images Plus 3.0 software (China).

Statistical analysis
Data for gene expression is represented as the mean ± standard deviation for every experimental group. Data for inflammation and H. pylori scores are represented with median values. The association between variables on an ordinal scale is presented by the Spearman rank correlation. Experimental data were compared among the groups using the Kruskal-Wallis test. p-values less than 0.05 were considered significant. ***p < 0.001. Data presented as mean ± SD of each experimental group. All data were analyzed using the GraphPad Prism 9 software.

Histopathological changes induced by H. pylori infection
In the present study, we used a mouse model of H. pyloriinduced gastritis to explore the relationship between the bacterium and the induction of proangiogenic and inflammatory mediators in vivo. We evaluated the histopathological changes and lesions induced by the H. pylori infection in the groups of challenged and unchallenged mice. Non infected mice showed a normal mucosal architecture (Figure 2A). Five weeks after colonization, H. pyloriinfected mice showed scattered immune cells infiltrating mucosa and submucosa, at antrum, corpus and the squamo-columnar transition. Two of the animals showed patches of mucous metaplasia in corpus ( Figure 2B). Ten weeks after challenge, mice showed minor immune cell infiltration at the mucosa, especially at the squamo-columnar and corpus-antrum transitions, with some mucous metaplastic patches ( Figure 2C). Twenty-week-colonized mice showed minor immune cell foci in antral mucosa, at the base of the corpus glands and squamo-columnar transition (not shown). Thirty weeks after inoculation, mice presented a stronger and widespread infiltration of immune cells in mucosa and submucosa of corpus and corpus-antrum transition, with hyperplasia and multiple foci of mucous metaplasia ( Figure 2D). Forty-and fiftyweek challenged animals also showed an abundant and extensive immune cell infiltration in mucosa and submucosa of the corpus, with large areas of hyperplasia and mucous metaplasia ( Figures 2E, F).

Description of the H. pylori colonization score on gastric mucosa
We assessed the H. pylori colonization status by culture and immunohistochemistry for each animal and found H. pyloripositive mice in all time points. In accordance with the bacterial score, the most robust infiltration of immune cells and more severe lesions were found at 30, 40 and 50 w.p.i ( Figures 2G,  H), p=0.0187).

H. pylori infection induces concomitant expression of Tnf-A, Angpt2 and VegfA
We assessed the expression at the mRNA and protein level of several proangiogenic factors in the gastric mucosa in our mouse model. In the H. pylori-infected animals Tnf-A, VegfA and Angpt2 mRNAs normalized to Gapdh showed an increased expression in time, showing the lowest DCt values at 30 to 50 w.p.i (Figures 3A-C). When comparing unchallenged animals with the 50 w.p.i. group, a significant upregulation was observed for Tnf-A, VegfA and Angpt2 mRNAs expression (Figures 3E-G)

Angpt1 mRNA exhibited a peculiar expression
In H. pylori-infected mice from 5 to 20 w.p.i., there was a slight increment that reached its top level of expression at 30 w.p.i., but at 40 and 50 w.p.i. the mRNA levels were drastically downregulated ( Figure 3D). At 50 w.p.i Angpt1 mRNA levels were significantly different from the not infected animals ( Figure 3H). When analyzing the expression of Tnf-A, VegfA, Angpt2 and Angpt1 altogether, it is worth nothing that Tnf-A, Vegf-A and Angpt2 mRNAs showed a significant upregulation starting at 30 w.p.i, that coincides with the downregulation of Angpt1 at the 40 and 50 w.p.i. This is important since all these molecules act in concert to trigger the angiogenic switch.
The Western blot analysis showed an upregulation of VegfA and Angpt2 proteins at 30 to 50 w.p.i ( Figures 3F, G, I). In contrast, Angpt1 protein was downregulated ( Figures 4F, H). Of note, two bands for Angpt2 protein with weights between 50-57 KDa, were detected in 30 and 40 w.p.i mice. This was not observed at 50 w.p.i. (Figure 3F).
We also performed immunohistochemistry to assess the expression pattern and localization of Angpt1 and Angpt2 in murine tissue. H. pylori infection induced Angpt2 expression in gastric epithelial cells. Unchallenged mice showed a strong staining for Angpt1 in blood vessels, but Angpt2 was not detected (Figures 4A, B). Angpt2 immunoreactivity was observed in gastric mucosa of infected mice, with an intense expression in metaplastic areas at the corpus mucosa in 30-and 50-w.p.i. challenged mice (Figures 4D-G), but no signal for Angpt1 ( Figure 4C).

Discussion
Pathological angiogenesis is a process that takes place during chronic inflammation and cancer. It involves activation of endothelial and immune cells that secrete pro-angiogenic factors such as VEGF-A, ANGPT2, TNF-a and MMPs, thus triggering proliferation of quiescent endothelium to make new blood vessels. This has been intensively studied in several cancer types, including GC. Although some evidence from in vitro models and studies in human gastric mucosa suggest that H. pylori infection may be involved in the induction of pro-angiogenic factors as VEGFA and HIF-1 (17- 19,[24][25][26], to the best of our knowledge, no studies have addressed the role of H. pylori infection in the induction of proangiogenic proteins as Angpt1 and Angpt2 in the gastric mucosa. Therefore, we used a mouse model of H. pylori-induced gastritis to explore the kinetics of the expression of Angpt1, Angpt2, Tnf-a and Vegf-A in vivo. We found a significant upregulation of Angpt2 mRNA and protein, whereas Angpt1 mRNA was downregulated. The ANGPT2/ ANGPT1 balance determines the fate of the endothelium. ANGPT2 has opposing role to ANGPT1 since it promotes blood vessel wall destabilization. This is achieved by competitively inhibiting the binding of ANGPT1 to Tie-2 and reducing Tie-2 activation and phosphorylation (1). ANGPT2 is produced by endothelial cells and macrophages, and plays a key role in the promotion of vessel sprouting, pericyte detachment and basement membrane remodeling. ANGPT1 is produced by pericytes and is associated with endothelium quiescence. ANGPT2/ANGPT1 imbalance parallels capillary destabilization; as the inflammatory response mounted against H. pylori infection persists, activated endothelial and infiltrating immune cells produce increasing ANGPT2 levels while ANGPT1 is downregulated, ultimately leading to vessel destabilization (1,6).
In mouse and human, the Angpt2/ANGPT2 gene encodes a 496 aa protein of 57 KDa. Hypoxia is an inducer of alternative splicing in cancer and endothelial cells that frequently display a temporal and tissue-specific expression (27). In humans, alternative splicing generates the ANGPT2 443 isoform lacking exon 2, which produces a smaller (443 aa) 51 KDa protein that is found in activated endothelial cells and macrophages. Reports indicate that functions as a more potent competitive inhibitor of ANGPT1 (8) and is upregulated in cancer cell lines, breast tumor tissues, and canine adrenocortical tumor tissue (27,28). In the performed Western blots we observed the presence of two bands with weights between 50-57 KDa for the infected mice of groups of 30 and 40 w.p.i. (Figure 3F), using an antibody (ab8452, Abcam) predicted to recognize both isoforms of Angpt2. We hypothesize that the observed bands could be Angpt2 443 . Nevertheless, as there are no previous reports for the expression of the isoform Angpt2 443 in mice in normal or pathological conditions, further transcriptomic and protein assays are necessary to check whether the observation of the present study corresponds to that isoform.
We observed a concomitant upregulation of Tnf-A, Vegf-A, Angpt2 mRNAs at 30, 40 and 50 w.p.i. In vitro studies have revealed that TNF-a upregulates the expression of VEGF-A, ANGPT2, and Tie2, and deregulates ANGPT1 genes at mRNA level (29,30). After translation, the ANGPT2 protein is stored in Weibel-Palade bodies at endothelial cells. The main factor causing its secretion is TNF-a, which ultimately results in autocrine inhibition of the Tie-2 receptor (31). TNF-a is mainly produced by activated macrophages and T lymphocytes, and is a potent activator of endothelium by inducing vasodilatation, increase of vascular permeability and the recruitment of immune cells (32). TNF-a expression is substantially elevated in response to H. pylori infection thus inhibiting gastric acid secretion, which in turn facilitates H. pylori survival (32). TNF-A transcription is induced by nuclear factor kappa B (NF-kB), which is activated in response H. pylori infection in gastric epithelial cells in vitro and in vivo (15,33,34), and upregulates VEGFR in endothelial cells (16).
Besides its role in cancer progression, angiogenesis plays a major role in the multi-step carcinogenesis process due to the fact that the gastric mucosa undergoes important histological changes, which may require gaining access to the vasculature in order to receive an adequate supply of nutrients and oxygen (35). Tuccillo et al. (36) found elevated levels of VEGFA in H. pylori-associated human gastritis. VEGFA and iNOS levels are also high in patients with chronic atrophic gastritis as well as in metaplastic and dysplastic areas (37). These observations, and ours, support the evidence pointing to the role of H. pylori as a promotor of angiogenesis.
The normal gastric epithelium does not express angiogenic factors; those are secreted by endothelial and some immune cells under stimulus. Several in vitro studies, including our own observations (manuscript submitted), show that in co-cultures H. pylori induces the expression of ANGPT2, VEGFA and other angiogenesis-related factors in gastric adenocarcinoma cell lines (17, 26,38). Atrophic, hyperplastic, metaplastic or dysplastic cells in H. pylori-infected gastric mucosa have accumulated genetic and epigenetic alterations that may change their gene expression programs thus leading to alteration in their phenotypes (39). In this context, it could be possible that the gastric epithelial cells themselves secrete some angiogenic factors, in response to signals from the inflammatory microenvironment, bacterial virulence factors (e.g. CagA, VacA, LPS, urease), or both ( Figure 5). In the present study we challenged mice with H. pylori SS1, which has a nonfunctional CagA protein. Therefore, the mechanisms by which the bacterium induces angiogenic responses may be CagAindependent. Other virulence factor, such as urease, may be implicated since it has been demonstrated that this enzyme induces ANGPT2 and other angiogenic factors in human gastric cell lines (17).
In conclusion, in this study we have found that H. pylori infection concurrently modulates the expression of several angiogenic mediators in the murine stomach. Specifically, this bacterial infection induces the expression of Angpt2, Tnf-A and VegfA at the mRNA and protein levels. Concomitantly, it downregulates Angpt1 expression in mouse gastric mucosa in a time-dependent manner. Nevertheless, our study has two major limitations; first, we had only one group of non-infected mice for the experiment that was euthanized at 50 weeks, but no control groups for early time points of infected animals. Hence, we show only the evolution of the expression in infected animals, and the comparison with the control group is shown only for the 50 w.p.i. mice. Second; the presented evidence describes the role of H. pylori in the modulation of the studied angiogenic factors, therefore, subsequent mechanistic and functional studies are necessary to establish the impact to H. pylori infection to angiogenesis in vivo.

Data availability statement
Datasets are available on request: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The animal study was reviewed and approved by Institutional Committee for Animal Care and Use of Animals (CICUA) of the University of Costa Rica (permission number CICUA-031-17).

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